1. Field of the Invention
The present invention relates to an optical system switching apparatus for a reflected fluorescence microscope.
2. Description of the Related Art
Generally, the illumination/observation optical system of a reflected fluorescence microscope has an arrangement as shown in FIG. 4. Referring to FIG. 4, illumination light from a light-projecting unit 17 having a light source 15 and a lens 16 for projecting light emitted from the light source 15, is emitted toward an excitation filter 2. The emerging illumination light (the optical axis of which is denoted by reference numeral 11) is subjected to wavelength selection by the excitation filter 2, and only illumination light having the selected wavelength is transmitted through the excitation filter 2. The illumination light (excitation light) which has been subjected to wavelength selection by the excitation filter 2 is further subjected to wavelength selection by a dichroic mirror 4, and selected illumination light is reflected by the dichroic mirror 4 downward, as shown in FIG. 4. The reflected light passes through an objective lens 18 along an observation optical axis 12 and reaches a sample surface 19. The dichroic mirror 4 is inclined at 45.degree. with respect to the illumination optical axis 11 and the observation optical axis 12.
The sample is impregnated with a fluorochrome or fluorescent pigment in advance. Thus, the sample surface 19 generates fluorescence when excited with excitation light. This fluorescence forms an image through the objective lens 18. While forming an image, the fluorescence is transmitted through the dichroic mirror 4 and subjected to wavelength selection by an absorption filter 3, so that only a fluorescence component having the selected wavelength is transmitted through the absorption filter 3. The fluorescence transmitted through the absorption filter 3 forms an image on an image formation plane 20 because of the operation of the objective lens 18. This image is observed with an observing section 21, e.g., an eyepiece or a television camera.
In actual observation, the wavelengths of optimum excitation light to be irradiated on the sample and of the fluorescence generated by the sample differ depending on the types of the fluorochromes to be used by the sample. Accordingly, the excitation filter 2, the absorption filter 3, and the dichroic mirror 4 must be switched as appropriate as required in accordance with the sample, i.e., the fluorochrome impregnated in the sample. An optical system switching apparatus 24 is generally used for this purpose.
FIG. 5 shows an example of an arrangement of the conventional optical system switching apparatus 24. This optical system switching apparatus 24 includes a turret 28 having a circular disk-like horizontal rest portion and a cylindrical supporting portion raised on the center of the rest portion through which a shaft 29 is coaxially inserted, and four cubes 25 (only two are shown in FIG. 5). The shaft 29 is fixed to a base 10 of the microscope body, so that the turret 28 is about a central axis 22 of the shaft 29. The four cubes 25 are concentrically arranged around the central axis 22 on the horizontal rest portion and detachably mounted on the turret 28.
Each cube 25 has a cube frame 1 consisting of two cube frame sections 1a and 1b fixed to each other with a screw 27. An excitation filter 2 is detachably mounted to a window of the cube frame section 1a with a set ring 5a. An absorption filter 3 is detachably mounted to a window of the cube frame section 1b with a set ring 5b. A dichroic mirror 4 is detachably mounted to the cube frame 1b with a leaf spring 6.
Four slidable projecting ridges 30 (only two are shown in FIG. 5) are formed on the outer circumferential surface of the cylindrical support portion of the turret 28 at an angular interval of 90.degree. to extend along the cylindrical support portion. A slide groove 31 is formed in one outer surface of each cube frame 1. The four cubes 25 are mounted to the turret 28 by fitting the slidable projecting ridges 30 in the corresponding slide grooves 31 and sliding the cubes 25 from above. The base 10 is threadably engaged with the lower end portion of the shaft 29 on which the turret 28 is rotatably mounted. The base 10 may be fixed to the microscope body in an optically desired position, thereby fixing the optical system switching apparatus 24 to the microscope body. Simultaneously, the reflecting surface of the dichroic mirror 4 is arranged at a position where a predetermined illumination optical axis 11 and an observation optical axis 12 intersect.
The operator can select a desired one of the four cubes 25 and arrange it in the illumination/observation optical path by touching a knurling portion 28a formed on the outer circumference of the horizontal rest portion of the turret 28 and rotating the turret 28 about the central axis 22 as the center. More specifically, an appropriate combination of an excitation filter 2, an absorption filter 3, and a dichroic mirror 4 can be used in the illumination/observation optical path in accordance with the fluorochrome impregnated in the sample.
Publications that disclose apparatuses of this type include Jpn. Pat. Appln. KOKOKU Publication No. 56-19605.
With the conventional optical system switching apparatus described above, however, although the combination of the excitation filter 2, the absorption filter 3, and the dichroic mirror 4 can be switched by selecting the cube 25, the excitation filter 2, the absorption filter 3, and the dichroic mirror 4 cannot be switched separately. For example, when observing samples colored with different fluorochromes, if the fluorescence components have different excitation band widths such as wide band width/narrow band width but close excitation spectra, only the excitation filter 2 need be exchanged in accordance with the excitation band width. In the conventional apparatus, however, since it is impossible to switch only the excitation filter 2, two cubes 25 are set to have the same absorption filters 3 and the same dichroic mirrors 4 while employing different types of excitation filters 2 having different band widths. Alternatively, the cubes 25 are removed from the turret 28 and disassembled, and the excitation filters 2 are exchanged for new ones.
Of the above cases, when two cubes 25 are to be prepared, two identical absorption filters 3 and two identical dichroic mirrors 4 must be prepared, and the number of types of absorption filters 3 and the number of types of dichroic mirrors 4 that can be used simultaneously by the entire switching apparatus are decreased to three each. It is also cumbersome to exchange the excitation filters 2.
One known observation method using a reflected fluorescence microscope is the ratio imaging method. The conventional optical system switching apparatus has a problem in executing this method as well. In the ratio imaging method, a fluorochrome whose the excitation spectrum changes as it is coupled with a material in the living body as the observation target is employed. This excitation spectrum is represented by excitation light having two specific wavelengths. The fluorescence intensities caused by this two-wavelength excitation light are measured, and the ratio in the fluorescence intensities is examined. A change over time of this ratio of the fluorescence intensities is regarded as a change in excitation spectrum of the fluorochrome, thereby indirectly obtaining a ratio with which the material in the living body is coupled with the fluorochrome. More specifically, when the fluorescence intensity is observed as an image, the ratio with which the material in the living body is coupled with the fluorochrome can be obtained. To know the coupled state of the material in the living body with the fluorochrome, a change over time of this image must be observed. For this purpose, an excitation filter 2 must be continuously switched to continuously change the excitation light described above between two wavelengths. In this case, since the number of light-measuring wavelengths having the fluorescence intensities selected by an absorption filter 3 is one, the absorption filter 3 need not be switched. Since the difference between the two wavelengths of the above excitation light is not very large, and an optical element that can reflect the two-wavelength excitation light is used as a dichroic mirror 4, the dichroic mirror 4 need not be switched.
Accordingly, of the excitation filter 2, the dichroic mirror 4, and the absorption filter 3, only the excitation filter 2 need be switched. In the conventional optical system switching apparatus, however, the excitation filter 2 can be switched only as a whole cube 25. As described above, in the ratio imaging method, as the excitation light must be continuously switched to observe a change over time, if the excitation filter 2 is switched by removing the cube 25, the switching operation cannot catch up with the change over time. Therefore, two excitation filters 2 of different types, two dichroic mirrors 4 of one type, and two absorption filters 3 of one type must be mounted to two cubes 25, and these cubes 25 must be switched. Therefore, two identical absorption filters 3 and two identical dichroic mirrors 4 must be used.
Furthermore, as the absorption filter 3 and the dichroic mirror 4 are also switched simultaneously when the excitation filter 2 is switched, an error occurs in the inclination or parallelism of the absorption filter 3 or dichroic mirror 4. As this adversely affects the observation optical axis, a positional error may occur in the image, so that the ratio of the fluorescence intensities may not be precisely measured in terms of position.
In recent years, fluorescence observation employing multi-color dyeing is widely performed. The conventional optical system switching apparatus has a problem in this respect as well. The filter characteristics and fluorescence spectrum characteristics of fluorescence observation employing two-color dyeing will be described with reference to FIG. 6. In FIG. 6, an ordinate represents the transmittance or fluorescence intensity of the filter 2 or 3, or the transmittance of the dichroic mirror 4, and an abscissa represents the wavelength. Two absorption filters a and b of different types will be used as absorption filters 3.
Referring to FIG. 6, reference symbol E.sub.X denotes the spectral transmittance curve of the excitation filter 2; D.M., the spectral transmittance curve of the dichroic mirror 4; E.sub.ma, the spectral transmittance curve of the absorption filter a to be described later; and E.sub.mb, the spectral transmittance curve of the absorption filter b to be described later. A curve FL indicates the intensity of the fluorescence generated by the sample.
In this observation method, only excitation light of the same wavelength indicated by the curve E.sub.X is transmitted through the excitation filter 2, and this excitation light is irradiated on the sample. The characteristics of the dichroic mirror 4 are indicated by the curve D.M. The dichroic mirror 4 does not transmit the excitation light therethrough but reflects it toward the sample.
The two types of fluorochromes impregnated in the samples are excited with excitation light having the same wavelength indicated by the curve E.sub.X to generate fluorescence. The respective fluorochromes have different fluorescence spectra. More specifically, the wavelength of the fluorescence generated by a fluorochrome C is mainly located near a peak FL.sub.c while the wavelength of the fluorescence generated by a fluorochrome D is mainly located near a peak FL.sub.d. Accordingly, to observe the fluorescence components generated by the fluorochromes C and D simultaneously, the absorption filter a (refer to the transmittance curve E.sub.ma) capable of transmitting both the peaks FL.sub.c and FL.sub.d is used, and to observe only the fluorescence component generated by the fluorochrome C, the absorption filter b (refer to the transmittance curve E.sub.mb) capable of transmitting only a wavelength near the peak FL.sub.c is used. As is apparent from the curves FL and D.M., the dichroic mirror 4 transmits fluorescence components of all the wavelength bands generated by the sample.
Therefore, in fluorescence observation employing multi-color dyeing, usually, the excitation filter 2 and the dichroic mirror 4 may be used unchanged while only the absorption filter 3 must be switched. In the conventional optical system switching apparatus, however, since switching can be performed only in units of cubes 25, two cubes 25 having the same excitation filters 2 and the same dichroic mirrors 4 but different absorption filters 3 must be prepared, and the cubes 25 must be switched. In this case, an inconvenience occurs similar to that which occurs when only the excitation filter 2 is to be switched, as described above.
Furthermore, recently, an absorption filter 3 and a dichroic mirror 4 both having a plurality of transmission bands have been developed. In exciting the fluorochromes of a sample dyed in multiple of colors, they enable observation of a fluorescent image having a high contrast through separation of fluorescence components caused by the respective fluorochromes, only by switching only the excitation filter 2 without switching the absorption filter 3 or the dichroic mirror 4. In this case, as the image does not cause a positional error due to switching of the absorption filter 3 or the dichroic mirror 4 described above, these absorption filter 3 and dichroic mirror 4 are preferable for photographing of a multi-exposure photograph or image analysis in accordance with multi-exposure with a television camera. In the conventional optical switching apparatus, however, the excitation filter 2 of the cube 25 must be switched, leading to a cumbersome operation.